Characterization of monoclonal antihuman small bowel and colon specific mucin antibodies.

Three human small bowel and colon mucosal specific monoclonal antibodies with distinct morphologic and electrophoretic characteristics were generated by fusion of immunized Balb/c spleen cells and murine plasmacytoma cells. Morphologic specificity by indirect immunofluorescence (IIF) revealed three antibody binding patterns corresponding to villus surface (TP-NG-43), goblet cell apical granules (TP-NG-2), and a combined surface/goblet cell apical granule antibody (TP-NG-20). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) produced three distinct electrophoretic migration patterns. These antibodies reacted with very high molecular weight determinants: TP-NG-2, one band greater than 400 kD; TP-NG-20, two bands corresponding to 370-400 kD; and TP-NG-43, two bands in the 350-400-kD range with smaller bands in the 50-94-kD range. Cross-reactivity with various other human organ systems was evaluated by indirect immunofluorescence and SDS-PAGE electrophoresis with Western blotting. By IIF, all three monoclonal antibodies reacted very strongly with components of gastric mucosa. Weak cross-reactivity was seen with colon, rectum and mucin-producing adenocarcinoma of the colon. No cross-reactivity was observed by IIF with other mucin-containing and non-mucin-containing tissues. However, cross-reactivity with gastric mucin was not detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Antibody reactivity with mucin was confirmed by purifying various regional gastrointestinal mucins and by subsequent testing by ELISA. Monoclonal antibody affinity columns were prepared and evaluated. The utility of these methods will allow for further definition of important goblet cell mucin glycoprotein characteristics and isolation of mucin subpopulations.

Genotyping of Bacteroides fragilis isolates from stool specimens by arbitrarily-primed-PCR.

In order to determine genetic relatedness of Bacteroides fragilis isolates from different clinical sources, arbitrarily primed polymerase chain reaction (PCR) (AP-PCR) was used to compare 17 strains isolated from patients with inflammatory bowel disease (IBD) and 20 strains isolated from foals with diarrhea. Three reference ATCC strains were also analyzed. Eighteen unique types were identified with a 22-mer arbitrary primer (ERIC-2) among the 20 patient isolates. Types 1 (enterotoxigenic) and 9 (nonenterotoxigenic), were each found in the stools of two patients. All other isolates showed a distinct and unique DNA banding pattern indicating a high degree of genotypic variability. Eleven types were identified among the foal isolates. Type 20, a nonenterotoxigenic type, was present in 30% of the foals. No correlation was found between the human and horse isolates. No clear relationship between a disease state (diarrhea or IBD) and specific types was observed. AP-PCR will be useful as a rapid method to determine genetic relatedness and in future epidemiologic studies of diarrheal diseases due to B. fragilis.

Sclerosing mesenteritis seen clinically as pancreatic pseudotumor: two cases and a review.

Sclerosing mesenteritis is an uncommon nonneoplastic inflammatory process in the mesentery that is seen as a pseudotumor, usually involving the small bowel mesentery, the mesenteric fat, and less commonly, the mesentery of the large bowel. We report two cases of sclerosing mesenteritis and review the literature on this rare disease. Both patients had pain, profound weight loss, and a mass on computed tomography (CT) scan of the abdomen. The provisional diagnosis was pancreatic neoplasm on the basis of clinical presentation and imaging studies. The diagnosis of sclerosing mesenteritis was established by histologic findings in biopsy material obtained at laparotomy in both cases. Interval histologic studies in one patient who had a high CA 19-9 level, progressive biliary ductal and partial duodenal compression, revealed a transitional histologic pattern from predominant inflammation and fat necrosis to predominant fibrosis. This may explain the varied descriptive terms used in the literature to describe this entity.

Microsporidial AIDS cholangiopathy due to Encephalitozoon intestinalis: case report and review.

Microsporidia are increasingly recognized as opportunistic infections in immunodeficient patients, predominantly patients with AIDS. The two microsporidia most commonly associated with disease in AIDS patients are Enterocytozoon bieneusi and Encephalitozoon intestinalis (previously known as Septata intestinalis). The most common clinical presentation of microsporidiosis in AIDS patients is diarrhea, most commonly caused by the Enterocytozoon bieneusi species. Encephalitozoon intestinalis is a recently described species that has been reported to cause disseminated human infection including cholangitis. We report a case of AIDS cholangiopathy that presented with abdominal pain and cholestatic liver tests. Ultrasound examination and ERCP revealed a picture of sclerosing cholangitis. Bile samples obtained at ERCP were negative for microsporidia; stool studies for microsporidia and cryptosporidia were also negative. No organisms were identified on routine light microscopy of the biopsy specimens from the duodenum, ampulla, and bile duct. E. intestinalis spores were demonstrated in the bile duct biopsies, by methylene blue and azure 11 staining and confirmed by electron microscopy. Albendazole therapy was successful in eradicating E. intestinalis with clinical improvement and improvement in CD4 count. However, the cholangiographic picture did not improve and repeat cholangiography revealed progressive bile duct injury. Albendazole therapy was delayed and may have been too late to prevent bile duct damage; the drug had to be approved by the US Food and Drug Administration for compassionate use. This is an unusual case of sclerosing cholangitis caused by an unusual organism and requiring biliary sphincterotomy and stent placement for progressive stricturing despite eradication of the infection.

Bacteroides fragilis enterotoxin gene sequences in patients with inflammatory bowel disease.

We identified enterotoxigenic Bacteroides fragilis in stool specimens of patients with inflammatory bowel disease and other gastrointestinal disorders. The organism was detected in 11 (13.2%) of 83 patients with inflammatory bowel disease. Of 57 patients with active disease, 19.3% were toxin positive; none of those with inactive disease had specimens positive for enterotoxigenic Bacteroides fragilis gene sequences.

Analysis of function, specificity and T cell receptor expression of cloned mucosal T cell lines in Crohn's disease.

Monoclonal populations of mucosal T cells were established from the earliest visible lesions in eight patients with well defined Crohn's disease. The FACS phenotype of all the mucosal derived clones to date are TCR alpha/beta+, CD3+, CD4+, and CD45RO+ memory cells. TCR variable region Beta chain analysis revealed predominantly V beta families 1, 2, 5.1, 5.2, 6, 7 and 8, with V beta family analysis supporting antigen expansion in the diseased mucosa. Putative autoreactivity was evaluated by stimulating individual clones with a battery of antigens and determining proliferation and IL-2 production by thymidine incorporation at 72 h. Antigens tested included crude Crohn's diseased (CD) colon and small bowel homogenates, CD brush border preparations, crude CD colon and small bowel mucin, and purified CD small bowel mucin. Controls included clone, APC, tetanus toxoid and either PHA or Staphylococcus enterotoxin B. A total of 200 clones were studied with 29.5% or 59 clones demonstrating proliferation and/or IL-2 production. T cell receptor V beta gene usage evaluated in a small number of reactive clones correlated with the expanded patient families. Seven of the fifteen represented families revealed diverse T cell receptor gene use and no disease overlap.

Are infectious agents involved in primary biliary cirrhosis? A PCR approach.

BACKGROUND/AIMS: A variety of data suggest that microbial infections and, in particular, atypical mycobacteria infections, may either initiate and/or be associated with the pathogenesis of primary biliary cirrhosis. METHODS: To address this hypothesis, use was made of polymerase chain reaction techniques and primers specific for the 16s rRNA gene of Eubacteria, Archaeabacteria, Mycobacteria and Helicobacter to determine if such sequences were detectable in liver tissue specimens from 29 patients with primary biliary cirrhosis. Similar liver tissues from patients with primary sclerosing cholangitis, chronic hepatitis, alcoholic liver disease and otherwise normal donors were analyzed in parallel. Genomic DNA was extracted from each of these liver tissue specimens using sterile techniques to avoid possible laboratory contamination. The DNA was subjected to polymerase chain reaction amplification using bacterial genus specific primers and the amplified products cloned and sequenced. Sequence data were analyzed by searching for homology to existing genes. RESULTS: Sequences from primary biliary cirrhosis and control livers corresponded to those found in a variety of bacteria, but no consensus sequence was found in primary biliary cirrhosis specimens. Neither Archaeabacteria nor Mycobacteria products were detected in liver specimens of patients with primary biliary cirrhosis, and Helicobacter pylori DNA was detected in only one primary biliary cirrhosis patient. CONCLUSIONS: Although bacterial infection, particularly with intracellular organisms, has been suggested to play a role in the initiation of primary biliary cirrhosis, there is no evidence from this study to suggest an ongoing chronic infectious process.